There is an urgent need to develop new drugs for the treatment of tuberculosis, both to shorten therapy so that higher completion rates can be achieved using fewer resources, and to address the growing problem of drug-resistant tuberculosis. Nearly all current antibiotics target essential genes or processes. Though new approaches that inhibit genes important for virulence may provide an alternative, targeting essential genes is likely to remain the predominant approach to develop new M. tuberculosis inhibitors. Characterization of essential genes whose function is unknown or incompletely understood, however, is challenging because they cannot simply be deleted. In the absence of null mutations, regulated conditional gene expression can be informative, but is difficult to achieve in M. tuberculosis using current tools. Technology derived from the clustered, regularly interspaced, short palindromic repeat (CRISPR) system of Streptococcus pyogenes, using the Cas9 endonuclease together with small RNAs to provide sequence specificity, has rapidly become a method of choice for gene editing in eukaryotes. A recent variation of this system, in which amino acid substitutions in Cas9 result in a protein that lacks nuclease activity but retains RNA and DNA binding (dCas9), has been used to obtain specific repression of gene expression in both bacteria and eukaryotes. The goal of this small RO3 project is to adapt this system for use in mycobacteria, particularly in M. tuberculosis. This goal will be achieved through two specific aims. First we will construct E. coli-mycobacterial shuttle vectors that incorporate anyhydrotetracycline-inducible gene expression to express dCas9 and the required small RNA constructs, to target genes for repression. Second we will test this system, using 1) a gene that expresses the fluorescent mCherry protein, so that repression can be readily quantified, and 2) two native chromosomal loci so that we can examine the function of this system under conditions in which it would be used. By targeting different sites in these genes we will be able to assess the extent to which different target binding sites, e.g. promoter region versus coding sequence, leads to repression of the gene of interest. Once developed, this system will be useful for investigators to characterize essential genes whose function is not known, and will facilitate the development of inhibitors that target the protein products of these genes.